Original Article

Journal of Inborn Errors of Metabolism & Screening 2017, Volume 5: 1–7 The Challenge of Diagnosis and Indication ª The Author(s) 2017 DOI: 10.1177/2326409816685735 for Treatment in Fabry Disease journals.sagepub.com/home/iem

Marco A. Curiati, MD1, Carolina S. Aranda, MD, MSc1, Sandra O. Kyosen, MD, MSc1, Patricia Varela, MSc2, Vanessa G. Pereira, PhD3, Vania D’Almeida, PhD3, Joa˜o B. Pesquero, PhD2, and Ana M. Martins, MD, PhD1

Abstract Fabry disease, caused by deficient alpha-galactosidase A lysosomal activity, remains challenging to health-care professionals. Laboratory diagnosis in males is carried out by determination of alpha-galactosidase A activity; for females, enzymatic activity determination fails to detect the disease in about two-thirds of the patients, and only the identification of a pathogenic mutation in the GLA gene allows for a definite diagnosis. The hurdle to be overcome in this field is to determine whether a mutation that has never been described determines a ‘‘classic’’ or ‘‘nonclassic’’ phenotype, because this will have an impact on the decision-making for treatment initiation. Besides the enzymatic determination and GLA gene mutation determination, researchers are still searching for a good biomarker, and it seems that plasma lyso-Gb3 is a useful tool that correlates to the degree of substrate storage in organs. The ideal time for treatment initiation for children and nonclassic phenotype remains unclear.

Keywords genotype-phenotype correlation, enzyme replacement therapy, alpha-galactosidase A deficiency, dried blood spot on filter paper, screening

Introduction a normal range of values, as found in healthy individuals.1,2,5–7 In those patients, a diagnostic is performed with GLA gene Fabry disease (FD) is an X-linked lysosomal storage disorder molecular analysis. caused by deficiency of alpha-galactosidase A (a-gal A) activ- Molecular analysis of the GLA gene is an essential diag- ity involved in the degradation of glycosphingolipids. Its defi- nostic tool in women or in patients who have very strong ciency leads to the accumulation of clinical suspicion due to the presented symptoms but (Gb3) inside the cells, causing several clinical abnormalities; the enzyme activity is within the normal range. Furthermore, the GLA gene is located on Xq22.1.1 it can inform the treating physician about the exact kind The clinical suspicion of FD begins with a selection of of mutation, which is generally related to the clinical characteristic signs and symptoms, such as acroparesthesia, , recurring headache, abdominal pain, chronic diarrhea, progressive loss of renal function, cardiomyopathy 1 generally associated with myocardial fibrosis, central nervous Reference Center in Inborn Errors of Metabolism, Universidade Federal de 1–4 Sa˜o Paulo, Sa˜o Paulo, Brazil system microangiopathy, and/or positive family history, or 2 Center for Research and Molecular Diagnosis of Genetic Diseases, also families with high prevalence of disease, cardio- Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil 1,2 myopathy, or ischemic encephalopathy. 3 Departament of Psychobiology, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, After diagnostic hypothesis of FD is raised, the diagnosis is Brazil made using laboratory testing. In males, the enzymatic activity Received October 09, 2016. Accepted for publication November 15, 2016. of a-gal A in plasma, leukocytes, skin biopsy, or dried blood spot (DBS) samples is measured. In females with clinical sus- Corresponding Author: Sandra O. Kyosen, Centro de Referenciaˆ em Erros Inatos do Metabolismo – picion, it is widely described in literature that there is a limita- CREIM, Rua Coronel Lisboa, Universidade Federal de Sa˜o Paulo, 957, CEP: tion in performing enzymatic activity assay, since women can 04020-041, Sa˜o Paulo, Brazil. range from almost undetectable, as can be found in males, up to Emails: [email protected]; [email protected]

This article is distributed under the terms of the Creative Commons Attribution 3.0 License (http://www.creativecommons.org/licenses/by/3.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). 2 Journal of Inborn Errors of Metabolism & Screening manifestations. Mutations known as ‘‘classic’’ have an earlier its metabolites (mainly lyso-Gb3) in urine/plasma and biopsies onset of symptoms, and usually cause a more severe clinical for histology/electron microscopy are also recommended.13 phenotype. On the other hand, ‘‘nonclassic’’ usually shows Lyso-Gb3 is similar to Gb3, only lacking the fatty acid slower disease progression with later onset of symptoms but chain.14 Levels of plasma lyso-Gb3 have been correlated with patients can also present severe organ damage.8 disease severity as well as with response to ERT.15,16 In male The treating physician has to be aware of the different kinds patients, lyso-Gb3 is a reliable diagnostic tool to distinguish of mutations and their clinical implications. Nonsense and fra- between classical and nonclassical FD from non-FD partici- meshift mutations are supposed to cause a severe phenotype, pants. However, in some cases of female patients, the levels since these mutations cause important damage to protein struc- of lyso-Gb3 overlap with controls, suggesting that increased ture (misfolding), leading to a highly dysfunctional protein.9 lyso-Gb3 values are very suggestive of FD, but normal values Mutations without previous description in literature require cannot exclude FD.17 Urinary lyso-Gb3 levels and its analogs further investigation not only by performing brain and are lower than plasma, but it has been demonstrated that male magnetic resonance imaging (MRI) but also by considering patients present higher levels of these markers when compared renal biopsy to evaluate which organs might be affected. There to females, and none of these markers are found in healthy are also controversial mutations, like the p.D313Y and intronic individuals.18 In addition, increases in lyso-Gb3/creatinine variants, described in literature as pathogenic by some ratio correlates with Gb3 concentration, types of GLA muta- authors10 and as benign by other authors.11 The treating phy- tion, gender, and ERT status,19 emphasizing the diagnostic sician should always rely on the patients’ clinical manifesta- value of this biomarker. tions and examination, rather than the genotype, when deciding whether to begin enzyme replacement therapy (ERT).12 Although there has been specific treatment over the last 15 Challenges in Biochemical Diagnosis years for FD, patients still face the challenge to have an early At our laboratory (LEIM-UNIFESP), DBS and leukocytes diagnosis that will allow a timely treatment initiation to avoid assays are performed to determine a-gal A activity through irreversible damage to organs. Treating physicians still have fluorometric methods using 4-methylumbelliferyl a-D-galacto- the challenge to decide what would be the ideal time to treat the pyranoside as substrate and a-N-acetylgalactosamine as later onset variants patients, and researchers of molecular a-galactosidase B inhibitor.20 These fluorometric assays are alterations face the challenge of the unknown effect of GLA the most commonly used for Fabry diagnosis, but the analysis variants. We will try to address some of these challenges. of a-gal A activity in DBS through tandem mass spectrometry has also become available.21–23 Since renal disease is a common feature among patients with Biochemical Diagnosis FD, individuals undergoing (with no other known diagnosis) have been considered a high-risk population for FD, Determination of a-gal A activity is usually the first laboratory with an overall prevalence of 0.62%, ranging from 0% to 1.2% test for diagnosing FD in index patients; if there is a known worldwide (reviewed by van der Tol et al).24 We performed a familial GLA mutation, a straightforward DNA analysis is indi- pilot screening protocol in which DBS samples from Brazilian cated. Many biological materials can be used for the analysis of hemodialysis centers were sent to LEIM-UNIFESP for a-gal A a-gal A activity, but the most common are plasma, leukocytes, activity analysis (this study was approved by the ethical fibroblasts, and DBS. The assay in leukocytes is regarded as the research committee from Universidade Federal de Sa˜o Paulo, ‘‘gold standard’’ and DBS assays are widely used for screening CEP #0384/05). We received 6277 DBS samples from male purposes. patients at hemodialysis centers located in all regions of Brazil, As an X-linked disorder, male patients can be reliably diag- and a-gal A activity was below the normal range in 320/6277 nosed with FD if presenting low or undetectable a-gal A activ- (5%) of samples. ity; results from DBS analysis below the normal range often As a primary screening protocol, patients with low a-gal A require a confirmatory test on leukocytes and, if low a-gal A activity in DBS were requested to send leukocyte samples to activity is also detected in this material, FD is confirmed. GLA confirm the diagnosis. From 320 patients with low enzyme sequencing for mutation identification is recommended for activity in DBS, only 176 (55%) actually sent new samples for index cases, which could provide important insights for prog- determination of a-gal A activity in leukocytes, probably due to nosis and treatment, as well as for genetic counseling but is not transportation issues, which is common in large countries like mandatory for diagnosis. Brazil—this type of sample must be transported under refrig- On the other hand, analysis of a-gal A activity in hetero- eration, in contrast to DBS, which can be transported at room zygous females is usually not conclusive for Fabry diagnosis, temperature or even sent by regular mail. From these, only as women may present normal enzyme levels in different sam- 20 (11.4%) showed a-gal A activity in leukocytes below the ples due to random X-inactivation. Therefore, in those cases, normal range. We then requested samples for molecular anal- identification of a pathogenic GLA mutation is mandatory to ysis to identify GLA mutations, but only 18 samples were define the diagnosis and carrier status of a female patient. received and mutations were identified in 8 patients. Thus, the Additional complementary tests such as measuring Gb3 and observed prevalence of FD among Brazilian hemodialysis Curiati et al 3 patients found in this pilot study was 0.1%. It is important to male neonates tested positive on enzyme assays and molecular note that 45% of patients with low a-gal A activity in DBS did tests,31 and 1:3024 Japanese neonates (both genders) also not send leukocyte samples and, regarding molecular analysis, tested positive on a-gal A activity assays and GLA gene anal- only GLA coding regions were analyzed in this study, which yses.32 The challenge in NBS for FD is that further studies are could explain why some patients presented low a-gal A activity necessary to determine the natural history of the later onset in leukocytes and had no mutations identified. Therefore, it is mutations to define the optimal timing for therapeutic possible that the observed FD prevalence may still be under- intervention. estimated (unpublished data). Timely treatment is very important to prevent irreversible The cutoff value of a-gal A activity in DBS used in this damage of target organs as shown in a study with 1044 adults screening protocol (2.5 mmol/L blood/h) was determined on the (641 men and 403 women) enrolled in the Fabry Registry, in basis of a prior validation of the DBS assay in a group of which many patients had advanced disease by the time of ERT healthy Brazilian volunteers.20 We decided to set a slightly initiation—among the patients who started ERT <40 years of high cutoff value to ensure that no patient with FD was missed age (n ¼ 526), 37% already had left ventricular hypertrophy in this initial analysis. However, since there was a high rate of (LVH) and 24% had an estimated glomerular filtration rate false positives in DBS, we decided to change our cutoff to 2.2 (eGFR) <60 mL/min/1.73 m2. When analyzing the population mmol/L blood/h after a receiver–operating characteristic curve that started ERT at 40 years of age or more (n ¼ 518), 33% had analysis, maintaining the assay sensitivity at 100%. In addition, an eGFR <60 mL/min/1.73 m2 and 79% LVH.33 another important alteration for the next screening protocol is In a systematic review of 20 case-finding studies, the overall that we will no longer request samples of leukocyte assays due prevalence of FD in men on dialysis was 0.33% and in women to the high transportation costs; patients with low a-gal A was 0.1%; the combined prevalence in renal transplant was activity in DBS will have DNA extracted from the DBS card 0.38% in men and 0% in women; in patients with LVH, the and sent directly to molecular analysis, which is described in overall prevalence ranged from 0.9% to 3.9% in men and 1.1% the next session. to 11.8% in women, depending on the selection of study pop- As quality control for FD diagnosis, another lysosomal ulation and study method.34 enzyme should be tested in the same DBS. The assay most Kitagawa and colleagues proposed that the measurement of commonly used for this purpose is evaluation of b-galactosi- GL-3 in urine by tandem mass spectrometry could be a reliable dase, which should be within the normal range to ensure sample screening method in hemizygotic patients.35 quality. Our reference range for b-galactosidase activity was We still have to overcome some challenges in screening or previously determined through analysis of healthy Brazilian case-finding studies, such as the fact that enzyme assay is not volunteers’ samples,20 and for a-gal A evaluation, only DBS suitable for screening females, thus the screening for mutations samples with b-galactosidase activity within the normal range on the GLA gene can be performed in this population; however, were used in this study. this is a laborious method because FD does not have common mutations and full sequencing of the GLA gene is necessary, and there are some variants that have unknown effect.24,36 Screening and Case Finding The FD has signs and symptoms peculiar in its clinical pre- sentation, generally poorly understood by physicians and mis- Molecular Diagnosis understood by them due to their lack of knowledge about this The human GLA gene is organized into 7 exons encompassing disorder; thus, it usually takes several years from the onset of over 12 kb; the exons range in length from 92 to 291 bp. There symptoms until the definite diagnosis is established, as is the are several possible regulatory elements in the 50-flanking case in most rare disorders. The timely diagnosis of FD remains region such as, ‘‘CCAAT’’ box, enhancer elements, and a challenge.25 In a study of 126 Brazilian patients enrolled in sequences corresponding to the activator protein 1, octanucleo- the Fabry Registry (61 males and 65 females), the median time tide, and ‘‘core’’ enhancer element but no typical ‘‘TATA’’ between the onset of symptoms and diagnosis was 20.3 years in box. Moreover, the GLA gene has an unmethylated CpG-rich males and 14.3 years in females; the median age of diagnosis island upstream of the initiation codon.37 The messenger RNA was 31.9 years in males and 27.1 years in females.26 encodes a protein of 429 amino acids, including the N-terminal Screening is defined as testing of apparently well people to signal peptide of 31 amino acids.37–39 After cleavage of the find those at increased risk of having a disease or disorder that signal peptide, posttranslational modifications occur in the should be medically important, with a known natural history Golgi and and a mature protein is formed.40 and an effective intervention must exist; looking for additional Mutations in the GLA gene have been identified using DNA illness in those with medical problems is termed case-finding, sequencing. The 7 exons of GLA gene are amplified by poly- and screening is limited to those apparently well.27 merase chain reaction. Sense and antisense oligonucleotide (NBS) for FD has gained some interest primers are synthesized based on the sequences flanking the since effective treatment became available.28,29 In NBS stud- 7 exons of the GLA gene. Sequences are compared with the ies, it was found that 1:3092 Italian male neonates had deficient reference sequence NG_007119 (http://www.ncbi.nih.gov) and a-gal A activities and specific mutations30; 1:2388 Taiwanese confirmed by reverse strand sequencing. To date, more than 4 Journal of Inborn Errors of Metabolism & Screening

840 mutations have been reported in the GLA gene in the enzyme and correlated genotype and phenotype by a meta- Human Gene Mutation Database,41 that are believed to cause analysis of the phenotypes reported in the literature.9 It appears FD. Of these, 589 are missense and nonsense mutations, 42 are that missense mutations that cause a mild phenotype affect splicing substitutions, 4 are regulatory substitutions, 114 are areas more distant from the enzyme active site. On the other small deletions, 40 are small insertions, 11 are small indels, hand, missense mutations associated with severe FD affect 35 are gross indels, 4 are gross insertions, and 6 are complex areas that are near the active site, leading to a more dysfunc- rearrangements. tional or inactive enzyme.9,48 In a study conducted in our laboratory with 568 individuals Nonsense mutations that lead to a premature stop codon from 102 families with suspected FD, we found 51 families usually result in an inactive protein or do not result in any presenting 38 different alterations in the GLA gene, among proteic product (‘‘null’’ allele). Missense mutations, on the which 19 were not previously reported in the literature.42 Most other hand, may severely reduce enzyme activity, but there mutations are detected in only 1 family, and it is necessary to may be some residual activity.49 Patients with residual determine the physiological impact of these new mutations. In enzyme activity have been described as exhibiting a later order to predict the possible impact of the new mutations on the onset of renal cardiac and neurovascular involvement, a structure and function of a human protein, different silico pre- decreased prevalence of neuropathic pain, and milder/later dictors are used. onset of other symptoms compared with those individuals In addition, in our sample we found an intronic haplotype with no residual enzyme activity.9,49 with 4 variants described in individuals with suspected FD. Several mutations are currently being reported as of unknown One variant, the c.-10C>T (rs2071225), was found in the significance, causing controversy in literature, showing that the untranslated region of exon 1. It is described in the literature diagnostic of FD in patients with nonspecific symptoms such as as polymorphism and appears in 12% of the population.43 (The LVH and a nonclassical phenotype can be difficult. Misdiag- single nucleotide polymorphism database [dbSNP] can be noses may occur in patients presenting with GLA mutations and found at http://www.ncbi.nlm.nih.gov/SNP.) The variants isolated nonspecific findings, such as LVH. In these cases, c.370-77_370-81delCAGCC (rs5903184), c.640-16A>G genetic and enzyme analyses are often not sufficient to diagnose (rs2071397), and c.1000-22C>T (rs2071228) are also found FD. A structured diagnostic approach is mandatory, including in intronic and are described in the literature as nonpathogenic electron microscopy of a biopsy of an affected organ.50 polymorphisms (dbSNP). Although these variants are Previous articles discuss the pathogenicity of a mutation in described as nonpathogenic, the impact of these changes an attempt not to misdiagnose a patient as having FD, starting a together is unknown. Considering that these regions are not very expensive treatment without needing it. routinely evaluated by gene sequencing, the prevalence of The D313Y genotype, previously reported in literature as a FD may be underestimated.44 benign mutation not clinically relevant toFD,11 has been reported in 2016 as having a significant impact on health- related quality of life in respective individual patients; this Challenges in GLA Gene Variants mutation might represent a cofounding risk factor for certain Most of the disease-causing mutations are de novo (private isolated symptoms triggering a specific mild clinical variant mutations) restricted to 1 single family or patient.1,2,45 Because of FD.51 of the frequency of novel mutations, it is necessary to sequence The p.A143T and p.R112H variants are other controversial the entire GLA gene and flanking regions to identify the FD mutations. The p.A143T variant has previously been reported mutation in a family. Also, as previously reported by Laney to be associated with a renal variant of FD.30 Later, Terryn et al et al in 2013, several familial mutations were identified by reported patients with the p.A143T variant as having no storage comparative genomic hybridization (CGH) array. They were in biopsies with light microscopy. They concluded that the caused by a microdeletion or microduplication in the GLA variant is most likely nonpathogenic. Additionally, individuals gene, where conventional genomic sequencing did not identify with the p.A143T variant have no increase in plasma Gb3 and a point mutation.46 lyso-Gb3.52 The genotype–phenotype correlation in FD is difficult to When facing a new or controversial mutation, patients pre- determine due to several causes. First, unlike other metabolic senting any symptomatology should be thoroughly investigated disorders, there are a high number of de novo mutations, to evaluate organic damage, performing brain and heart MRI, implying that the majority of families carry different muta- nephrologic evaluation, and laboratory assessment. In cases of tions.9,47 Another cause that impedes the correlation is the single-organ impairment, a targeted biopsy showing signs of clinical variability among patients carrying the same muta- deposit can confirm or discard the diagnostic and should be tion, even among patients in the same family. Recently, it has performed in order to correctly indicate ERT. been hypothesized that part of the phenotype can be modified by nongenetic features, such as accumulation of the misfolded defective enzyme.9 Indications for Treatment In 2004, Garman and Garboczi48 mapped GLA mutations Many questions about the optimal time to initiate FD treatment onto a crystallographic model of the structure of a-gal A are still present in clinical practice. The main studies guide the Curiati et al 5 physician who has the autonomy of therapeutic decision as he nonclassic disease, the current recommendation is frequent or she often already knows the family history and the compli- monitoring with different medical specialties. That treatment cations involved. should be promptly initiated when symptoms emerge or in the Information about the evolution of the disease is not always presence of an abnormal renal biopsy.59 available, so some suggestions can be applied. First, the differ- entiation according to gender should be made by the team that Conclusion treats a patient with FD. Another feature to be considered is whether the disease presents itself in classical or nonclassical The challenges in the diagnosis and indications for treatment of form, as described above. In most consensus and expert opi- FD are part of today’s clinical practice. Thus far, we have nions on FD, those with classical or nonclassical disease should learned that the enzymatic assay is diagnostic for men, while be treated as soon as the first target organ impairment signals women most often need the molecular study to have a definite are noted, such as in the kidney, heart, or central nervous sys- diagnosis. The enzyme activity assay in DBS has its established tem.6,53–55 In Brazil, for all ages greater than or equal to 18, use in case studies in high-risk populations due to the stability treatment should be indicated if the following are present: of the enzyme in this kind of sample and ease of shipping. The effects of controversial mutations in exons or multiple intronic microalbuminuria, , or renal failure, are not yet fully understood and seem to have individual varia- cardiac hypertrophy, even without signs of fibrosis or tions. In this situation, the disease staging of the patient will cardiac signals (such as sinus bradycardia or give us the answer if the ERT is indicated or not. We believe cardiac repolarization modifications), today that treatment is indicated in children with acroparesthe- white matter lesions or transient ischemic attack, sia due to the relationship that seems to exist with their pres- gastrointestinal symptoms (GIS) with chronic diar- ence and renal impairment. In conclusion, FD is still being rhea or abdominal pain (GIS may be due to neuro- studied and in the medium-/long-term, we will have some pathic and myopathy changes leading to symptoms of answers and probably more questions. dysmotility56), or acroparesthesia, even if controlled with , since Declaration of Conflicting Interests the neuropathic pain is associated with glycosphingoli- The author(s) declared no potential conflicts of interest with respect to pid accumulation in small fiber neuropathy, and the the research, authorship, and/or publication of this article. combination of acroparesthesia and mild glomerular endothelial cell deposits and arteriopathy may constitute Funding a clinical and morphological combination heralding a The author(s) received no financial support for the research, author- potentially progressive renal disease.57,58 ship, and/or publication of this article.

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